This grant focuses on the underlying molecular mechanisms of the ability of Drosophila melanogaster to tolerate very low O2 levels for prolonged periods without any functional or morphological evidence of injury. Using the Drosophila model system, mutants obtained from a genetic screen (X-Ray/EMS and P-elements), gene over-expression using the Gal-4/UAS with EP lines and a new and exciting direction of inducing more resistant flies with hypoxia exposure over generations, we have obtained very promising and exciting preliminary data. Since it has become clear that many biological fundamental pathways are conserved during evolution, we believe that studies on Drosophila have become very relevant to human biology, behavior and disease. Our general hypothesis is that low O2 induces cellular alterations via pathways that are shared by other processes that may not be related to hypoxia. In this application we have the following specific hypotheses and aims: A) Drosophila melanogaster tolerance to hypoxia is based on the presence of specific genes, most of which are fundamental and are not only important in hypoxia tolerance but also in other types of stresses (e.g, oxidant injury, CO2). Our aims include i. the use of a P-element screen to continue with the forward genetic approach;ii. the use of secondary phenotypic assays (e.g. CO2) when hypoxia-sensitive mutants are identified and B) In spite of the fact that Drosophila is very tolerant to low O2 levels, there are genes that, if mutated or over-expressed, will enhance the resistance of flies to hypoxia;this will be determined using various complementary approaches and techniques. Our aims include the use of hypoxia (chronic, over generations) as a selection pressure to develop more tolerant flies;and ii. characterize functionally (mutations, over-expression) the genes obtained. We believe that one of the major strengths of this application is that we are not only trying to determine the genes that seem to be relevant for anoxia tolerance but we also examine the function of genes cloned in the context of cell and whole body physiology. Our hope is that, by 1) collaborating with experts in various areas important for our proposal, and 2) understanding the mechanisms underlying hypoxia tolerance, we can manipulate hypoxia-sensitive cells (e.g., mammalian) to render them resistant